Agr typing of Staphylococcus aureus species isolated from clinical samples in training hospitals of Isfahan and Shahrekord.

OBJECTIVE: As an opportunistic pathogen, Staphylococcus aureus is associated with serious nosocomial infections and growing antimicrobial resistance against beta-lactams among S. aureus strains has become a global challenge. The current study was designed to investigate the presence of agr genes among S. aureus strains recovered from clinical samples in university hospitals of Isfahan and Shahrekord.
RESULTS: A total of 150 S. aureus isolates were screened by Disk diffusion method (DDM) and conventional PCR. The minimum (17.3%) and maximum (46%) antibiotic resistance rates were found in vancomycin and cefoxitin, respectively. The majority of our isolates were classified as agr type I followed by type II, type IV, and type III. The statistical analysis showed a significant correlation between agr type I and antibiotic resistance against cefoxitin and erythromycin (p = 0.04 and p = 0.03, respectively). Based on our findings, the agr typing could be considered an effective approach for molecular tracking of S. aureus infections.

Introduction

As a part of microflora of skin and mucous membranes of healthy individuals, Staphylococcus aureus is also an opportunistic pathogen and associated with hospital acquired infections such as septicemia, pneumonia, septic arthritis, osteomyelitis, toxic shock syndrome after surgery, folliculitis, endocarditis, and urinary tract infections (UTIs) [1, 2]. Antibiotic resistance by affecting more than two million people annually is one of the biggest global challenges. The increasing antimicrobial resistance among S. aureus species against beta-lactam antibiotics has led to serious problems with the treatment of their related infections. Despite considerable efforts in controlling antibiotic resistance, methicillin-resistance S. aureus (MRSA) is raising worldwide, in addition geographical and local variations influence its dynamic and crisis [1, 3]. The methicillin resistance development in S. aureus is related to the several Staphylococcal Cassette chromosome mec elements (SCCmec) encoding mecA gene for a penicillin binding protein (PBP2a) [4]. MRSA strains are usually multi-drug resistant (MDR) and show resistancy to other antibiotics such tetracyclines, aminoglycosides, lincosamides etc. [1, 5, 6]. Rapid and precise typing of S. aureus is really crucial to transmission identification of this pathogen. In this regard, Pulsed-Field gel electrophoresis and spa typing (Staphylococcal protein A) are common typing methods. The spa gene is one of the most distinctive factors related to this organism, and various patterns of it have been identified by several studies [7]. One of the major regulatory and control factors in the virulence gene expression of S. aureus is the accessory gene regulatory (agr) system. Indeed, agr operon including agrA, agrB, agrC, and agrD genes regulate over 70 genes in S. aureus 23 of which control its pathogenicity and invasive infections [8]. Moreover, S. aureus can be stratified into 4 different groups (agr I, agr II, agr III, and agr IV) according to the sequences of agrC (auto inducing peptide) and agrD (cyclic AIP) genes. It is stated that agr types are different in their properties and prevalence in various geographical areas thus, identification of predominant types in each region may well be functional [9].

Given to the critical roles of agr genes, the current study was designed to detect and identify the agr groups of S. aureus strains isolated from clinical samples in training hospitals of Isfahan and Shahrekord cities.

Main text

Materials and methods

Samples and bacterial isolates

This cross sectional study was conducted in microbiology department of Shahrekord University of Medical Sciences. During May to November 2017, a total of 150 isolates of S. aureus were collected from clinical samples (wound, blood, urine, tissue etc.) of patients attending university hospitals in Isfahan (Alzahra and Kashani) and Shahrekord (Kashani and Hajar).

Characterization assays

The isolates were identified using Gram staining, catalase test, slide or tube coagulase test, DNase test, and growth on Mannitol Salt Agar (MSA) as a differential growth medium [10].

Antibiogram testing

Disk diffusion method (DDM) as described by CLSI 2016 guideline [11] was performed for following antibiotics: erythromycin (15 mg), tetracycline (30 mg), vancomycin (30 mg), gentamicin (10 mg), rifampin (5 mg), cefoxitin (15 mg), trimethoprim (5 mg), rifampicin (5 mg). In addition, all isolates were subjected to cefoxitin disc diffusion test to identify the methicillin sensitive S. aureus (MSSA) and MRSA.

DNA extraction

The nucleic acids of S. aureus isolates were extracted by phenol chloroform method followed by RNase treatment [12]. The purity of extraction was assessed using the A260/280 ratio and agarose gel electrophoresis.

PCR amplification of the mecA gene

molecular detection of mecA gene was carried out according to the following condition: initial denaturation at 95 °C for 3 min followed by 33 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C for 30 s, and extension at 72 °C for 1 min and final extension step at 72 °C for 6 min.

PCR detection of agr genes

PCR assay for amplification of agr genes was set as follows: hot start at 95 °C/6 min, 32 cycles of 94 °C/45 s, 60 °C/1 min, 72 °C/70 s and a final extension step of 72 °C/8 min. All reactions performed in duplicate and along with the negative control (water) and positive (previously known positive-PCR products) control. The final products were detected by electrophoresis on 1% agarose gel containing DNA safe stain (Sinagene, Iran) and the sizes of the PCR products were estimated by the migration pattern of a 100-bp DNA ladder (Sinagene, Iran).

Statistical analysis

Statistical analysis was performed using SPSS version 22. The chi-square test was used to calculate statistical significance (p < 0.05).

Results

Study population

150 S. aureus isolates were collected from patients attending training hospitals in Isfahan (110 isolates from Alzahra hospital) and Shahrekord (25 cases from Kashani hospital and15 isolates from Hajar hospital). The mean age of the participants was 47.6 years (SD: 21.5) and male/female ratio was 90/60. However, there was not any significant difference in sex and age of patients with S. aureus infection. S. aureus isolates were obtained from several clinical samples and different hospital wards.

Antibiotic susceptibility

According to our results the lowest (17.3%) and the highest (46%) antibiotic resistance rates were found in vancomycin and cefoxitin, respectively. In addition, MRSA strains were verified by PCR amplification of mecA gene. The antibiotic resistance distribution among different agr groups is shown in Table 1. The results of this study showed a significant correlation between agr type and antibiotic resistance against cefoxitin and erythromycin (p = 0.04 and p = 0.03, respectively).

Table 1

The antibiotic resistance profiles among 4 different agr types

AGR type	FOX		E			T			VA		RP			TM			GM
	S (%)	R (%)	S (%)	I (%)	R (%)	S (%)	I (%)	R (%)	S (%)	R (%)	S (%)	I (%)	R (%)	S (%)	I (%)	R (%)	S (%)	I (%)	R (%)
I	37 (45.1%)	45 (54.9%)	38 (46.3%)	21 (25.6%)	23 (28)	47 (57.3%)	7 (8.5%)	28 (34.1%)	70 (85.4%)	12 (14.6%)	49 (59.8%)	3 (3.7%)	30 (36.6%)	52 (63.4%)	6 (7.3%)	24 (29.3%)	58 (70.7%)	2 (2.4%)	22 (26.8%)
II	22 (59.5%)	15 (40.5%)	12 (32.4%)	5 (13.5%)	20 (54.1%)	20 (54.1%)	1 (2.7%)	16 (43.2%)	28 (75.7%)	9 (24.3%)	27 (73.0%)	2 (5.4%)	8 (21.6%)	18 (48.6%)	5 (13.5%)	14 (37.8%)	23 (62.2%)	0	14 (37.8%)
III	7 (70%)	3 (30%)	3 (30%)	1 (10%)	6 (60%)	7 (70%)	0	30 (30%)	8 (80%)	2 (20%)	8 (80%)	1 (10%)	1 (10%)	4 (40%)	1 (10%)	5 (50%)	7 (70%)	1 (10%)	2 (2%)
IV	15 (71.4%)	6 (28.6%)	6 (28.6%)	7 (33.3%)	8 (38.1%)	10 (47.6%)	1 (4.8%)	10 (47.6%)	18 (85.7%)	3 (14.3%)	14 (66.7%)	1 (4.8%)	6 (28.6%)	14 (66.7%)	1 (4.8%)	6 (28.6%)	13 (61.9%)	0	8 (38.1%)
Total	81 (54%)	69 (46%)	59 (39.3%)	34 (22.7%)	57 (38%)	84 (56%)	9 (6%)	57 (38%)	124 (82.7%)	26 (17.3%%)	98 (65.3%)	7 (4.7%)	45 (30%)	88 (58.7%)	13 (8.7%)	49 (32.7%)	101 (67.3%)	3 (2%)	46 (30.7%)

agr typing

Molecular detection of 150 S. aureus isolates has indicated that agr type I was the predominant one (82/150) followed by type II (37/150), type IV (21/150), and type III (10/150) (Fig. 1). Table 2 is shown the frequency distribution of different agr types among different clinical samples.

Fig. 1

Lanes 1 and 2: agr type III, lanes 3 and 4: agr type II, lane 5: agr type I, lane 6: agr type IV, M: 100 bp DNA ladder, C− and C+: negative and positive controls

Table 2

Frequency distribution of agr types in different clinical samples

Sample	agr type				Total
	I	II	III	IV
Tracheal aspirate
No	13	6	2	2	23
%	56.5%	26.1%	8.7%	8.7%	100%
Wound
No	30	11	4	5	50
%	60.0%	22.0%	8.0%	10.0%	100%
Tissue
No	3	2	0	0	5
%	60.0%	40.0%	0.0%	0.0%	100%
Abscess
No	5	5	1	1	12
%	41.7%	41.7%	8.3%	8.3%	100%
Conjunctival swab
No	0	1	0	1	2
%	0.0%	50.0%	0.0%	50.0%	100%
Blood culture
No	1	2	0	0	3
%	33.3%	66.7%	0.0%	0.0%	100%
Sputum
No	2	0	0	2	4
%	50.0%	0.0%	0.0%	50.0%	100%
Nasal aspirate
No	2	1	1	0	4
%	50.0%	25.0%	25.0%	0.0%
Synovial fluid
No	0	0	0	3	3
%	0.0%	0.0%	0.0%	100%	100%
Discharge
No	3	3	1	0	7
%	42.9%	42.9%	14.3%	0.0%	100%
Bactace
No	7	1	0	2	10
%	70.0%	10.0%	0.0%	20.0%	100%
Blood
No	4	3	0	1	8
%	50.0%	37.5%	0.0%	12.5%	100%
Peritoneal fluid
No	1	0	1	0	2
%	50.0%	0.0%	50.0%	0.0%	100%
Urine culture
No	9	2	0	3	14
%	64.3%	14.3%	0.0%	21.4%	100%
Pharyngeal swab
No	2	0	0	1	3
%	66.7%	0.0%	0.0%	33.3%	100%
Total
No	82	37	10	21	150
%	54.7%	24.7%	6.7%	14.0%	100%

Discussion

There is a dramatic increase in S. aureus infections, both with community-associated and hospital-acquired types, and development of antibiotic-resistant species, especially MRSA and vancomycin-resistant strains, is the major cause of the infections and further treatment complications [13]. Identification and typing of the isolates may imply a common source of infection; therefore, accurate analysis of these patterns can help to break the chain of transmission. Accordingly, the present study was designed to identification of agr types among S. aureus isolates and possible association of these pathogens with some phenotypic characteristics such as antibiotic resistance and pathogenesis.

Dufour et al. [14] used agr typing method for the first time to stratify S. aureus isolates and affirmed that these bacteria can be divided into four groups I, II, III, IV by this system. Ever since, many researches have been applied the agr typing approach and in several studies such as those by Lee et al. and Shopsin et al. [15, 16], the agr group I was the most dominant S. aureus type. Our findings indicated that agr type I was the most predominant type among S. aureus isolated from Isfahan and Shahrekord cities. Similarly, in several previous studies such as those by Cheraghi, Bibalan, Peerayeh, Khoramrooz, Mohsenzadeh, and Goudarzi agr type I has been reported as the most dominant isolate of S. aureus in different regions of Iran [17-22]. It is declared that certain agr groups of S. aureus are involved in some particular disease and infections, for example agr type I isolates are associated with bacteremia and invasive infections [21]. In the present study, wound and tracheal aspirates, were sequentially the most frequent clinical samples and as it is summarized in Table 2, the agr group I was the major agr type among these sample. However, we couldn't find any significant difference or correlation between agr types and certain clinical specimen.

In the current study, the antimicrobial susceptibility testing revealed that the highest antibiotic resistance rate was against cefoxitin (46%) followed by erythromycin and tetracycline (both 38%). Several studies that have reported erythromycin and tetracycline as the antimicrobial agents with lowest susceptibility among S. aureusagr group I isolates [20, 23, 24]. As it is summarized in Table 1, the agr types III and I showed the maximum and minimum resistance rates against tetracycline, respectively. In the present study, agr type I isolates had the highest sensitivity to vancomycin; however, the smallest resistance rate against this agent was related to agr type IV (Table 1). The greatest susceptibility and resistance to rifampin were found among agr types IV and I of S. aureus strains, respectively (Table 1). As it could be seen in Table 1, agr types IV and III have shown the highest and the lowest susceptibility to trimethoprim, respectively. The maximum percentage of gentamicin susceptibility was related to agr type I, while type III isolates had the highest resistance against this antimicrobial agent (Table 1). We found a significant correlation between agr type and antibiotic resistance against cefoxitin and erythromycin (p = 0.04 and p = 0.03, respectively). Indeed, in this report, the agr types never implied the sensitivity or resistance to antibiotics, but in the case of cefoxitin and erythromycin the agr group I isolates showed the highest resistance against these agents.

The majority of S. aureus isolates in this study were classified as agr group I and our results suggest a probable correlation between this type and antibiotic resistance to cefoxitin and erythromycin. Here we can conclude that agr typing is a suitable and effective approach for molecular tracking of S. aureus infection.

Limitations

The lack of investigation on others typing methods in S. aureus isolates can be mentioned as one of the main limitations of the present study.